Method of producing β-cyclodextrin

ABSTRACT

The present invention; is a method of producing β-cyclodextrin at a higher efficiency from a raw material other than starch, comprising making a malto-oligosaccharide with 2 to 10 glucoses co-exist with cyclodextrin glucanotransferase in a solution containing an organic solvent which can precipitate 50% or more of β-cyclodextrin when an excessive amount of the solvent is added to the solution of the β-cyclodextrin and effecting the reaction at a lower temperature than 40° C.

TECHNICAL FIELD

The present invention relates to a method of producing cyclodextrin.More specifically, the present invention relates to a method ofproducing β-cyclodextrin at a higher efficiency from a raw materialexcluding starch.

BACKGROUND ART

Cyclodextrin; hereinafter refered to as "CD", is a nonreducingmalto-oligosaccharide where 6 to 12 glucoses are bonded together in acyclic form via glucoside bonding. For industrial CD production, α-CDwith six glucoses bonded together, β-CD with seven glucoses bondedtogether, and γ-CD with eight glucoses bonded together, are produced asa single product or a mixture thereof.

CD, an amphoteric substance having the hydrophilic outer ringcircumference and also having the hydrophobic pore, has an inclusionfunction to stably include a variety of molecules and the like in thehydrophobic pore.

Therefore, CD potentially prepares volatile matters into non-volatilematters or provides masking of unpleasant odor, so CD is applicable in awide variety of fields, for example, for use in medicinal products,foodstuffs, cosmetics and the like.

CD has conventionally been produced via an enzyme reaction, ie. thereaction of a CD generating enzyme (cyclodextrin glucanotransferase;hereinafter referred to as "CGTase") with starch as a substrate. Thesubstrate concentration should preferably be higher from industrialpoint, which involves the increase in the viscosity of the reactionsolution whereby the stirring procedure gets hard along with a lowerreaction rate. Thus, a method has been proposed, comprisingprelimlnarily subjecting starch to the pretreatment process withα-amylase for liquidation to decrease the viscosity, but the process iscomplex.

Because the reaction product is produced as a mixture of CDs of α, β,and γ type, purification is needed so as to obtain β-CD for primary use,and thus, the decrease of the yield is unavoidable. Alternatively, noreport has been issued of the CD production via CGTase using a substrateother than starch.

The present invention has been undertaken from such respect. Theobjective of tile present invention is to provide a method of producingβ-CD with a substrate other than starch at a higher efficiency.

DISCLOSURE OF THE INVENTION

The present inventors have made intensive investigations so as toachieve the above objective. Consequently, the inventors have found thatthe production of β-CD alone can be achieved by using amalto-oligosaccharide of a higher concentration as a raw materialsubstrate followed by the reaction with CGTase.

That is, the present invention is a method of producing β-cyclodextrincomprising steps of making a malto-oligosaccharide with 2 to 10 glucosesco-exist with cyclodextrin glucanotransferase in a solution containingan organic solvent which can precipitate 50% or more of β-cyclodextrinwhen an excessive amount of the solvent is added to the solution of theβ-cyclodextrin and effecting the reaction at a lower temperature than40° C.

The present invention will now be explained in details.

The process of producing the CD in accordance with the present inventionis characterized in that CGTase effects on such malto-oligosaccharide inthe presence of the organic solvent.

Such malto-oligosaccharide as the raw material substrate is preferablythose of a polymerization degree of 2 to 10, and these may be usedsingly or in a mixture thereof. Maltose is specifically preferable interms of ready availability and production cost.

As CGTase, use is made of the enzymes which are generated from Bacillusmacerans, Bacillus megaterium, Bacillus circulans and the like and whichare to be used generally for industrial CD production. However, theseenzymes catalyze various reactions, in accordance with the presentinvention, β-CD is generated via intramolecular conversion (cyclization)or intermolecular conversion (disproportionation).

Preferably, such organic solvent forms an inclusion compound togetherwith β-CD relatively strongly, illustratively including one of cyclichydrocarbons such as cyclohexane, cyclooctane, cyclododecane and thelike, aromatic hydrocarbons such as benzene, ethyl benzene, o-xylene,m-xylene, p-xylene, o-dichlorobenzene, naphthalene, anthracene and thelike, halogen compounds such as tetrachloroethylene, chloroform and thelike; or a mixture of two or more thereof. Among then, cyclohexane isspecifically preferable.

The organic solvent in the reaction solution is preferably at aconcentration of 30 to 80 vol %, more preferably 40 to 60 vol %, and ismixed and stirred with water or a buffer for the reaction.

When maltose is used as the substrate, the yield of β-CD reaches maximumwhen the substrate concentration is at 40 w/v % and it does not increaseeven if the concentration is more than 40 w/v %, so the synthesis shouldpreferably be done in a range below the concentration in terms of yield.Similarly, the yield moves toward maximum around 60 hours after theinitiation of the reaction. When the reaction period is far longer than60 hours, the yield is lowered. Thus, the reaction should be carried outfor 60 hours or less. Also, pH of the reaction solution is preferably4.5 to 8, more preferably 5 to 7.

The optimum temperature of CGTase is around 50 C, but if the reaction iscarried out at a temperature above 40° C., CD is not generated.Therefore, the reaction should be carried out at a temperature lowerthan 40° C., preferably at 25° C. or less, more preferably at 10° C. orless.

Because the generated β-CD precipitates after the formation of aninclusion compound together with the organic solvent, the β-CD canreadily be separated from the reaction solution and unreacted substrate.

When CGTase effects on a malto-oligosaccharide, themalto-oligosaccharide is modified via intramolecular conversion into amalto-oligosaccharide of nearly glucose monomer to glucose octamer(malto-octanose). Herein, an oligosaccharide of not less than glucosehexamer is possibly modified into CD via intramolecular conversion.Because the malto-oligosaccharide is also present simultaneously in thesystem, however, the generated CD is rapidly degraded via ring-openingconversion so that almost no CD remains in the system.

If the reaction is carried out in a system with the organic solventadded to generate the precipitate of the reaction product after theformation of an inclusion compound with CD, the CD generated viacyclization is hardly exposed to the enzyme action and is thereforeaccumulated in the reaction system.

In accordance with the present invention, furthermore, CD is generatedvia the reaction at 40° C. or less, which is supposed due to the factthat an inclusion compound of CD with an organic solvent is present inmore stable manner when the temperature is set at 40° C. or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the chromatograms of the products by β-CDsynthesis; in FIG. 1A cyclohexane was not added; in FIG. 1B cyclohexanewas added.

FIGS. 2A and 2B show the chromatograms of the products by β-CD synthesisafter glucoamylase treatment; in FIG. 1A cyclohexane was not added; inFIG. 1B, cyclohexane was added.

FIG. 3 is a graph depicting the relation between the amount ofcyclohexane in the reaction solution and the yield of β-CD.

FIG. 4 is a graph depicting the relation between the cyclohexaneconcentration and the relative yield of β-CD.

FIG. 5 is a graph depicting the relation between the substrateconcentration and the yield of β-CD.

FIG. 6 is a graph depicting the effect of pH on the synthesis of β-CD.

FIG. 7 is a graph depicting the relation between the yield of β-CD andthe reaction time.

FIG. 8 is a graph depicting the relation between the yield of β-CD andthe reaction temperature.

BEST MODE FOR CARRYING OUT THE INVENTION

The examples of the present invention will now be described in details.In the following examples, the CGTase (product name; Contizyme derivedfrom Bacillus macerans, commercially available from AmanoPharmaceuticals, Co. Ltd.) was used at 300 U (in Tilden-Hudson Unit: J.Bacteriol. 43, 527(1942)) per gram substrate. The enzyme primarilygenerates α-CD when starch is employed as the substrate.

For the analysis of the reaction products, use was made of glucoamylase,derived from Rizopus niveus and commercially available from SeikagakuKogyo, Co. Ltd.

EXAMPLE 1

Explanation will follow of CD production in a cyclohexane-water system,using maltose as a substrate.

CD synthesis was performed in 0.5 ml of 50 mM acetate buffer (pH 5.2)with various amount of cyclohexane. Under stirring at 7.5° C., thereaction was continued at a 20% substrate concentration for 66 hours.

The reaction products were analyzed by HPLC (high-performance liquidchromatography) on a column, ASAHIPAK NH2P-50 (manufactured by AsahiKasel, Co. Ltd.).

FIG. 1 shows the chromatograms of the reaction products with nocyclohexane added (A) and with 0.4 ml of cyclohexane added (B). At a17.857-min position of B, a peak was observed, which was not observed inA. The peak has the same retention time as that of the standard β-CD.

FIG. 2 shows the chromatograms of the reaction products afterglucoamylase was added to these products to decompose linearmalto-oligosaccharide. Herein, CD is not decomposed with glucoamylase.The peak corresponding to the standard β-CD remains in D. These resultsindicate that the peak is that of β-CD.

FIG. 3 shows the calculated yield (%) of β-CD in the individualreactions when the peak area described above was used as an indicator.FIG. 4 shows the yield of β-CD relative to the cyclohexane concentration(cyclohexane volume % to the total volume of the reaction solution) whenthe maximum yield of β-CD was defined as 1.0.

These results indicate that β-CD is generated, by using maltose as thesubstrate through the reaction of CGTase in a cyclohexane-water system.Also, it is indicated that the cyclohexane concentration is preferably30 to 80%, more preferably 40 to 60%.

EXAMPLE 2

Examination was then done about solvents to be used in accordance withthe present invention. To 1% solutions each of α-CD, β-CD and γ-CD, anorganic solvent was added at an excess amount followed by stirring forseveral minutes at room temperature for HPLC analysis of the supernatantof the aqueous phase so as to determine the amount of the precipitate.The solvent in which α-CD, β-CD or γ-CD was absolutely precipitated wasdesignated as a solvent of 100% precipitation ratio. Those solventswhich were solid at room temperature were dissolved in hexane for use.

As in Example 1, similar reactions were effected when each of thesolvents was adjusted to a concentration of 44.4%. The analysis was doneof the presence or absence of precipitate during the reaction and of theprecipitate per se, if any. The results are shown in Table 1.

                  TABLE 1    ______________________________________               Precipitation ratio                             Precipitate    Organic solvent    CD           α β  γ                                       during reaction    ______________________________________    Hexane       39.2    50.9    17.9  x    Decane       49.0    14.1    11.6  x    Dodecane     60.7    25.8    17.1  x    Tridecane    62.8    16.2    17.2  x    Hexadecane   69.5    19.2    17.8  x    C Dlohexane  84.1    94.0    0     β    C Dlooctane  8.7     56.4    20.0  β    C Dzene      11.1    87.5    11.8  β    Ethyl benzene    C D          13.3    93.3    94.9  β    C Dylene     0       97.7    96.6  β    C Dylene     0       93.8    88.6  β    C Dylene     10.1    96.7    27.0  β    C Dichloro-  5.3     98.9    99.2  β    benzene    C Drachloro- 8.0     98.4    93.9  β    ethylene    C Doroform   2.1     89.5    92.2  β    C Dlodo-     35.7    91.0    0     β    decane +    Hexane    Naphthalene +    C D          55.4    99.5    85.5  β    Hexane    C Dhracene + 34.0    90.8    0     β    Hexane    ______________________________________

These results indicate that β-CD can be generated by the use of anorganic solvent capable of precipitating about 50% or more of β-CD.

EXAMPLE 3

β-CD was synthesized under various concentrations of substrate. Theenzyme was maintained at a constant level (300 U/g) relative to theweight of the substrate. Cyclohexane (44.4%) was used as an organicsolvent, for carrying out the reaction for 48 hours under the sameconditions as in Example 1. The results are shown in FIG. 5.

The results indicate that the elevation of the maltose concentrationincreases the yield of β-CD. However, the yield reaches plateau when theconcentration is above 30%.

EXAMPLE 4

Examination was done about the effect of pH on the β-CD synthesis usingas a substrate maltose (20%). Cyclohexane (44.4%) was used as an organicsolvent; as a buffer, 50 mM acetate buffer was used at pH 4 to 5 whileat pH 6 to 8, 50 mM phosphate buffer was used. The results are shown inFIG. 6.

These results indicate that the preferable pH is pH 5 to 8.

EXAMPLE 5

Furthermore, examination was done about the relation between thereaction time and the yield of β-CD in the β-CD synthesis.

Using maltose (20%) as a substrate, the reaction was effected at 7.5° C.in a mixture of 30 ml of cyclohexane and 24 ml of 100 mM acetate buffer(pH 6.0). The results are shown in FIG. 7.

The results indicate that the CD yield reached maximum around 60 hoursafter the initiation from the reaction, and that the yield was graduallylowered thereafter.

EXAMPLE 6

The effect of temperature on the β-CD synthesis was examined. Using as asubstrate maltose (20%) and as an organic solvent cyclohexane (44.4%),the reaction was effected in a mixture of 50 mM phosphate buffer and theorganic solvent at various temperatures for 48 hours. The results areshown in FIG. 8.

The results indicate that no synthesis of β-CD occurred at 40° C. butthat such reaction occurred at a temperature less than 40° C.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, the production of β-CD fromthe raw material other than starch is realized. In accordance with thepresent method, the viscosity of the reaction solution is not elevatedeven when the raw material is at a higher concentration. Thus, nopretreatment thereof is required. Because the reaction product is β-CDalone, the purification procedure is simple.

We claim:
 1. A method of producing β-cyclodextrin, comprising the steps of:enzymatically converting a substrate consisting essentially of maltose into β-cyclodextrin, using cyclodextrin glucanotransferase, in a solution containing 30 to 80 vol % of an organic solvent, at a temperature lower than 40° C., at a pH and for a time sufficient to generate β-cyclodextrin, said organic solvent capable of precipitating 50% or more of β-cyclodextrin if added to a solution containing β-cyclodextrin, wherein only β-cyclodextrin is substantially generated and precipitated in the solution such that any generated β-cyclodextrin is substantially degraded in the absence of said solvent; and recovering the precipitated β-cyclodextrin from the solution.
 2. A method according to claim 1, wherein said solution contains 40 to 60 vol % of the organic solvent.
 3. A method according to claim 1, wherein said pH is in the range of 5-8.
 4. A method of producing β-cyclodextrin, comprising the steps of:enzymatically converting a substrate of malto-oligosaccharide into β-cyclodextrin, using cyclodextrin glucanotransferase, in a solution containing 30 to 80 vol % of an organic solvent, at a temperature of 10° C. or lower, at a pH and for a time sufficient to generate β-cyclodextrin, said organic solvent capable of precipitating 50% or more of β-cyclodextrin if added to a solution containing β-cyclodextrin, said malto-oligosaccharide having 2 to 10 glucose monomers such that any generated β-cyclodextrin is substantially degraded in the absence of said solvent in the absence of said organic solvent, wherein only β-cyclodextrin is substantially generated and precipitated in the solution; and recovering the precipitated β-cyclodextrin from the solution.
 5. A method according to claim 4, wherein said malto-oligosaccharide is maltose.
 6. A method according to claim 1 or 5, wherein the organic solvent is selected from the group consisting of cyclohexane, cyclooctane, benzene, ethyl benzene, o-xylene, m-xylene, p-xylene, o-dichlorobenzene, tetrachloroethylene, chloroform, cyclododecane, naphthalene and anthracene. 